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. Author manuscript; available in PMC: 2020 May 18.
Published in final edited form as: Mol Plant Microbe Interact. 2020 Mar 3;33(4):569–572. doi: 10.1094/MPMI-11-19-0324-A

Genome Sequence of Fusarium oxysporum f. sp. matthiolae, a Brassicaceae Pathogen

Houlin Yu 1,3, Dilay Hazal Ayhan 2,3, Andrew C Diener 4,†, Li-Jun Ma 1,2,3,†
PMCID: PMC7233306  NIHMSID: NIHMS1587034  PMID: 31967942

Abstract

The filamentous fungus Fusarium oxysporum is a soil-borne pathogen of many cultivated species and an opportunistic pathogen of humans. F. oxysporum f. sp. matthiolae is one of three formae speciales that are pathogenic to crucifers, including Arabidopsis thaliana, a premier model for plant molecular biology and genetics. Here, we report a genome assembly of F. oxysporum f. sp. matthiolae strain PHW726, generated using a combination of PacBio and Illumina sequencing technologies. The genome assembly presented here should facilitate in-depth investigation of F. oxysporum-Arabidopsis interactions and shed light on the genetics of fungal pathogenesis and plant immunity.

Keywords: genome, Fusarium oxysporum f. sp. matthiolae, Arabidopsis thaliana, model, microbe-plant molecular interaction

Genome Announcement

Strains of the filamentous fungus Fusarium oxysporum, a notorious plant pathogen, can infect hundreds of cultivated species and at the same time have distinct host-specificity (Kistler 1997; Michielse and Rep 2009; Ma et al. 2013). This host-specificity is used to classify F. oxysporum into formae speciales, and a forma specialis typically represents one to a few monophyletic clonal lineages that cause disease in a narrow range of taxonomically related plants (Kistler 1997). At the genomic level, host-specificity corresponds to the presence of lineage-specific chromosomes (Ma et al. 2010). However, little is known about molecular mechanism involved in these host-specific plant-fungal interactions. One of the three formae speciales that are pathogenic to the crucifer Arabidopsis thaliana (Diener and Ausubel 2005; Provart et al. 2016), the genome sequence of F. oxysporum forma specialis matthiolae will enable the genetic analysis of fungal pathogenesis and host immunity using the model plant Arabidopsis thaliana.

Phylogenic analyses indicate that Fom isolates form a single clonal lineage (Bosland and Williams 1987; O’Donnell et al. 2009; Kistler and Benny 1989; Kistler et al. 1987; Kistler et al. 1991), although two races of Fom are distinguished by the differential susceptibility of varieties of M. incana (Bosland and Williams 1988). Natural variation of immunity is observed among wild accessions or ecotypes of A. thaliana toward Fusarium wilt (Diener and Ausubel 2005). Quantitative trait loci (QTLs) mapping in offspring of crosses between resistant and susceptible ecotypes has identified three RESISTANCE TO F. OXYSPORUM (RFO) genes, one receptor-like protein gene (RFO2) and two receptor-like kinase (RLK) genes (RFO1 and RFO3) from different RLK gene subfamilies (Diener and Ausubel 2005; Diener 2013; Shen and Diener 2013; Cole and Diener 2013). As receptor-mediated immunity is reported to be the major determinant of disease resistance to Fom (Cole and Diener 2013), investigation of the interaction of Fom and A. thaliana should lead to a fundamental understanding of receptor-mediated plant immunity, especially against fungal pathogens. The genome sequence described here comes from DNA purified from Fom race 2, isolated from wilted garden stock (Matthiola incana), a cultivated plant in the crucifer or mustard (Brassicaceae) family, prized for its colorful flowers (Baker 1948; Tatsuzawa et al. 2012). This strain was previously deposited in American Type Culture Collection (ATCC 16603) by GM Armstrong and subsequently designated by PH Williams as PHW726 (Kistler et al. 1987).

The pipeline for genome assembly was adapted from Ayhan et al. 2018. Genomic DNA was purified from the mycelium of PHW726, and then sequenced by Illumina MiSeq and PacBio RS II platforms with 119× and 21× coverage, respectively. We used MiSeq paired-end sequencing with 150 cycles. The maximum size of the PacBio RS II reads was 59 kb while the mean size was 8.5 kb. Trimmomatic version 0.32 (Bolger et al. 2014) was used to remove adaptors and trim ends of Illumina reads (parameters: ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36). FastQC (version 0.11.5) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to check the quality of all reads. SPAdes version 3.9.1 (Antipov et al. 2016, using default parameters) was used to combine PacBio subreads and trimmed Illumina reads into an initial hybrid assembly. BWA version 0.7.12 (Li and Durbin 2009) was used to map the Illumina reads to the assembly. Further cleaning, fixing, and sorting of mapping reads was done with Picard version 2.0.1 (http://broadinstitute.github.io/picard/) and Samtools version 1.3 (Li et al. 2009). A structural variant (SV) caller, GRIDSS version 1.4.1 (Cameron et al. 2017) was used to identify links between scaffolds in the initial assembly. A custom script (available at github.com/dayhan/tools/scaffolding.m) was used for scaffolding. Minimap2 version 2.17 (Li 2018) was used to map PacBio subreads to new scaffolds, and links were manually inspected and, if necessary, fixed. Further polishing was performed by re-mapping Illumina reads to the assembly, during which FreeBayes v0.9.10–3-g47a713e (Garrison and Marth 2012) was used to identify base variants between reads and the assembly (specially, 70% support of minimal 10 alternate counts, with a minimal base mapping-quality greater than q30). Identified variants were used to correct the assembly by a custom script (available at github.com/d-ayhan/tools/FASTAeditWithVCF.m). RepeatMasker 4.0.5 (Tarailo-Graovac and Chen 2009) was used to screen the repeats. Mummer 3.22 (Kurtz et al. 2004) was used to align the assembly with the reference genome assembly for the tomato pathogen F. oxysporum f. sp. lycopersici Fol4287 (Ma et al. 2010).

As summarized in Table 1, the final assembly was 57.3 Mb in total length and comprised of 583 scaffolds with an N50 value of 0.77 Mb. The largest scaffold size was 3.6 Mb. The GC content was 47.4%. The size of total interspersed repeats was 3.1 Mb, which accounted for 5.4% of the assembly. A comparison with Fol4287 assembly (Ayhan et al. 2018) suggested a larger assembly size and higher interspersed repeat content of PHW726. The size of sequence mapped to the core chromosomes of Fol4287, which including 66 scaffolds (defined as core scaffolds), was 43.8 Mb. The assembly also included a scaffold of 52,365 bp that captured the entire mitochondrial DNA. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession WJXY00000000. The version described in this paper is version WJXY01000000. This assembly for the genome of PHW726 should facilitate future molecular genetics and genomic studies. Candidate Fom genes that promote pathogenesis or elicit immune response in A. thaliana and M. incana can now be predicted, subcloned and genetically characterized.

Table 1.

Summary of the F. oxysporum f. sp. matthiolae strain PHW726 genome assembly and a comparison with Fol4287 (Ayhan et al. 2018)

Variables Statistics
PHW726 Fol4287
Assembly size (bp) 57,270,650 53,912,367
Core sequence size (bp) 43,818,233 42,239,438
Number of scaffolds 583 499
Number of core scaffolds 66 55
Size of largest scaffold (bp) 3,557,637 5,733,288
Interspersed repeat content 5.35% 4.21%
N50 (bp) 774,050 1,338,693
N90 (bp) 47,752 49,310
L50 (bp) 18 11
GC content 47.44% 47.68%
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Acknowledgements

We thank Dr. Ravi Ranjan at the UMass Amherst Genomic Center and Dr. Quan Zeng, at the Connecticut Agricultural Experiment Station, for their consistent support for our genome projects, and the MGHPCC for providing high-performance computing capacity for the genome assembly process.

Funding Resources

This work was supported by the National Science Foundation (IOS-1652641). D.H.A. and L.-J.M. are also supported by the Burroughs Welcome Foundation (grant 1014893) and National Institute of Health (NIH/NEI R01 EY030150).

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